Duchenne Muscular Dystrophy (DMD), the most common inherited muscular dystrophy, leads to progressive muscle weakness and death by the third decade of life. A conundrum is that the mouse model that has the same genetic defect, absence of dystrophin, does not mimic the human disease, which has limited the development of efficacious therapies. Recently, we hypothesized that telomere length differences between mice and humans could account for this discrepancy and developed a mouse model that lacks dystrophin and has shortened telomeres (mdx/mTRKO ). This model exhibits all of the major pathological hallmarks of human DMD. In particular, the mdx/mTRKO mice exhibit impaired regeneration and progressive muscle wasting due to functional defects in their muscle stem cells (MuSCs). Here we address a major challenge: MuSCs are known to be functionally heterogeneous, but the nature of their diversity has yet to be characterized which is essential for targeting therapies. We propose to capitalize on two groundbreaking technologies developed in our laboratories to elucidate the defects in the signaling networks that underlie the dysfunctional MuSC subsets in a manner previously not possible. To this end we will use novel (1) state-of-the-art single cell mass cytometry (CyTOF) and (2) artificial bioengineered stem cell niches. The CyTOF is uniquely suited to identify dysfunctional MuSC subsets. Using new parameters identified by CyTOF, we will purify these subsets and in conjunction with our biomimetic hydrogel microwell platform perform single-cell fate mapping. The combination of highly multivariate single cell CyTOF analyses and real time single cell time-lapse imaging will reveal dysfunctional signaling profiles and behaviors within subsets of stem cells. Defects in signaling pathways identified in specific MuSC subsets in the mouse model will be validated in MuSCs isolated from human DMD patient samples. These subsets and pathways can then be targeted, leading to novel therapeutic strategies that will enhance muscle fiber repair in DMD patients.